MicroRNA-877-5p promotes osteoblast differentiation by targeting EIF4G2 expression

Stimulating bone formation potentially suggests therapeutics for orthopedic diseases including osteoporosis and osteoarthritis. Osteoblasts are key to bone remodeling because they act as the only bone-forming cells. miR-877-5p has a chondrocyte-improving function in osteoarthritis, but its effect on osteoblast differentiation is unknown. Here, miR-877-5p-mediated osteoblast differentiation was studied. Real-time reverse transcriptase-polymerase chain reaction was performed to measure miR-877-5p expression during the osteogenic differentiation of MC3T3-E1 cells. Osteoblast markers, including alkaline phosphatase (ALP), collagen type I a1 chain, and osteopontin, were measured and detected by alizarin red staining and ALP staining. Potential targets of miR-877-5p were predicted from three different algorithms: starBase (http://starbase.sysu.edu.cn/), PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), and miRanda (http://www.microrna.org/microrna/home.do). It was further verified by dual luciferase reporter gene assay. The experimental results found that miR-877-5p was upregulated during the osteogenic differentiation of MC3T3-E1 cells. Overexpression of miR-877-5p promoted osteogenic differentiation, which was characterized by increased cell mineralization, ALP activity, and osteogenesis-related gene expression. Knockdown of miR-877-5p produced the opposite result. Dual luciferase reporter gene assay showed that miR-877-5p directly targeted eukaryotic translation initiation factor 4γ2 (EIF4G2). Overexpression of EIF4G2 inhibited osteogenic differentiation and reversed the promoting effect of overexpression of miR-135-5p on osteogenic differentiation. These results indicate that miR-877-5p might have a therapeutic application related to its promotion of bone formation through targeting EIF4G2. Supplementary Information The online version contains supplementary material available at 10.1186/s13018-023-04396-y.


Introduction
Osteoporosis is a globally prevalent bone condition connected with bone resorption and loss of the bone microstructure, which can lead to bone fragility and increased bone fracture [1].One of the key methods to manage the imbalance in bone mass is to stimulate osteoblast formation [2].Osteoblasts, as the main cell type for bone formation, are the key to the metabolic homeostasis, growth, and injury repair of bone tissue [3][4][5], and some protein markers, such as alkaline phosphatase (ALP), collagen type I a1 chain (COL1A1), and osteopontin (OPN), are believed to have value as bone development biomarkers [6].Therefore, investigation into the regulatory Osteoblast differentiation is closely regulated by a variety of factors [7][8][9], among which miRNAs have recently received attention.miRNAs exert their regulatory functions by regulating their target genes at the post-transcriptional level, thereby participating in various biological processes [10][11][12][13][14]. miR-25 [15], miR-33-5p [16], miR-221, and miR-15b [17] have been revealed to act on corresponding targets, thus affecting the balance between bone formation and resorption, and therein regulating osteoblast differentiation.miR-877-5p is frequently downregulated in various cancers [18][19][20], and particularly, miR-877-5p has an alleviating impact on osteoarthritis chondrocytes [21].In addition, miR-877-3p promotes osteoblast differentiation of MC3T3-E1 cells by targeting Smad7 [22].However, the role of miR-877-3p in osteoblast differentiation has not been studied.
Therefore, this study aimed to investigate the role of miR-877-5p and EIF4G2 in osteoblast differentiation and found that miR-877-5p promoted osteoblast differentiation through targeted regulation of EIF4G2 expression.Our results may be useful in enhancing new bone formation and designing treatments for pathological bone loss.

RNA quantification
Total RNA was extracted from cells using TRIzol reagent (Thermo fisher, USA).The purity and concentration of RNA were spectrophotometrically analyzed using a Nanodrop One (Thermo Fisher).To determine miRNA, cDNA was transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher).To determine mRNAs, cDNA was transcribed using the First Strand cDNA Synthesis Kit (Beyotime, China).PCR was performed on an ABI 7500 Real-Time PCR system (Thermo Fisher) using the TB Green Premix Ex Taq II (Taraka, Japan).U6 was the reference gene for miRNA in the cell, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the reference gene for mRNA in the cell.The gene expressions were quantified using the 2 −ΔΔCT method.Sangong (Shanghai, China) was commissioned to synthesize primers (Table 1).

ALP staining and alizarin red staining
For ALP staining, after induction for 14 days, cells were fixed with 95% ethanol and stained with BCIP/NBT solution according to the manufacturer's protocol (Beyotime Institute of Biotechnology) at room temperature for 2 h.The ALP-positive cells were stained blue/purple.Stained cells were visualized using the Canon IXUS210 camera (Canon, Inc.; magnification).In addition, an Alkaline Phosphatase Staining Kit (BestBio, Inc.) was used to detect the ALP activity, according to the manufacturer's protocol.
For alizarin red staining, after induction for 14 days, cells were washed one or two times with phosphatebuffered saline (PBS), fixed with 95% ethanol for 10 min, washed one or two times with PBS again, covered, and stained with 0.1% alizarin red solution for 10 min.Finally, they were rinsed with PBS and observed under an inverted light microscope.For quantification analysis, 10% hexadecyl pyridinium chloride monohydrate (CPC; Sigma-Aldrich; Merck KGaA) was used to dissolve the mineralized nodules and then the absorbance was measured at 540 nm using a Multiskan ™ FC spectrophotometer (Thermo Fisher Scientific, Inc.).

Dual luciferase activity assay
The amplified EIF4G2 fragment containing the miR-877-5p binding site was cloned into the psi-CHEK2 vector (Promega) to obtain the EIF4G2 wild-type (WT).Also, an EIF4G2 mutant (MUT) vector was produced.MC3T3-E1 cells co-transfected with the above plasmids and miR-877-5p mimic or NC using Lipofectamine 2000 (Invitrogen).Luciferase activity was determined using a dual luciferase reporter assay system (Promega).

Statistical analysis
Statistical analysis was performed using PRISM v5.0 software (GraphPad, La Jolla, CA, USA).All data in three replicates are expressed as mean standard deviation and assessed by Wilcoxon test.P < 0.05 was considered statistically significant.

Suppressive effects of EIF4G2 on osteoblast differentiation
Transfection with si-EIF4G2 or oe-EIF4G2 was performed before osteoblast differentiation induction, and the vectors were successfully transfected (Fig. 4A).

Discussion
Osteoblast differentiation is an essential component of bone remodeling to maintain bone health [26].Inappropriate regulation of bone formation is associated with diseases such as osteoporosis, osteoarthritis, and bone cancer.The osteoblast differentiation process involves the regulation of gene expression, osteoblast differentiation marker genes, and mineral deposition [7][8][9].Currently, many miRNAs emphasize great roles in skeletal development and homeostasis, especially in osteoblast differentiation [27][28][29].Therefore, our study investigated miR-877-5p in osteoblast differentiation from the perspective of gene regulation and concluded that miR-877-5p promotes osteoblast differentiation by negatively regulating EIF4G2 expression.This is the first demonstration of the role and potential mechanism of miR-877-5p in osteogenesis.MC3T3-E1 osteoblast cell line can be used as a host for in vitro studies of bone remodeling and formation [30][31][32][33] due to its pre-osteoblast phenotype, and its subclone 14 can mineralize the collagen extracellular matrix [34].Therefore, mouse osteogenic MC3T3-E1 cells were applied to induce osteoblast differentiation model.Numerous studies have shown that miRNAs could act as key modulators in osteoblastic differentiation.MiR-497-5p stimulates osteoblast differentiation through HMGA2-mediated JNK pathway [30].MiR-135-5p promotes osteoblast differentiation by targeting HIF1AN in MC3T3-E1 cells [35].In addition, miR-532-3p inhibits osteogenic differentiation in MC3T3-E1 cells by downregulating ETS1 [36].In this study, it was found that miR-877-5p was upregulated during the osteogenic differentiation of MC3T3-E1.Osteogenesis-related markers in osteoblast differentiation, including OPN, COLIA1, and ALP, are at high levels during extracellular matrix maturation and mineralization [37,38].Furthermore, mineralization of the extracellular matrix represents the end stage of osteoblast differentiation and is considered a sign of osteoblast maturation [39,40].Here, we found that miR-877-5p upregulation increased the mineralization of MC3T3-E1 cells and induced ALP activity and osteoblast markers' expression.
Nonetheless, the results are only applicable to in vitro osteoblast models and it is unclear whether miR-877-5p and EIF4G2 have similar effects in animal models.Since bone formation can be mediated by the recruitment of mesenchymal stem cells [47], the process of MSC differentiation into osteoblasts should be further explored.

Conclusion
We reveal for the novelty that miR-877-5p promotes osteoblast differentiation by targeting and negatively regulating EIF4G2 expression.Therefore, therapeutic strategies targeting miR-877-5p may promote bone formation and may be effective in treating orthopedic diseases.